Lyme disease (sometimes referred to herein as LD or Lyme borreliosis) is a common vector-borne disease that is a significant public health concern. The disease is transmitted by the bite of various species of Ixodes ticks carrying the etiologic agent, a pathogenic Borrelia bacterium (a spirochete). Organisms of the Borrelia burgdorferi sensu lato group belong to the family Spirochaetaceae, genus Borrelia. There are at least 11 species in the B. burgdorferi complex and an unknown but large number of substrains. At least three genospecies of the Borrelia burgdorferi sensu lato group have been identified as pathogens: B. burgdorferi sensu stricto, B. afzelli, and B. garinii. In addition, other species of Borrelia have been implicated as being causative pathogenic agents. The major reservoir of the infection in the United States is the white footed mouse, and the infection can be transmitted to many mammalian species, including various other forms of wildlife, e.g. Eastern chipmunks, and dogs, cats, and humans.
Clinically, Lyme disease is a progressive disease with a wide array of manifestations. Early diagnosis and treatment is critical to prevent progression. Late disseminated infection can be associated with permanent damage to the nervous and musculoskeletal systems. Unlike most bacterial diseases that can be defined microbiologically by direct observation or culture of the pathogen, B. burgdorferi is difficult to culture or observe in clinical samples. Therefore, Lyme disease is defined indirectly. Erythema migrans (EM) is the classic marker for this infection at early stages. However, not all patients infected with pathogenic Borrelia develop EM. In the absence of EM, the current basis for diagnosis is the demonstration of an antibody response against a pathogenic Borrelia in an appropriate clinical setting.
Unfortunately, current serologic assays for such antibodies suffer from both low sensitivity and specificity, especially in early disease. The U.S. Centers for Disease Control and Prevention (CDC) currently recommends that in order for a patient to be considered seropositive, two assays must be positive: a first tier assay, such as an ELISA, IFA or lateral flow assay, followed by a second tier assay, such as a western blot. This approach is expensive and can delay diagnosis for a week or more, but it is necessary because of the poor specificity of the most commonly used first tier assays. There is a need for a simple, sensitive and specific diagnostic method for the detection of Lyme disease, particularly at early times after infection.
A peptide-based immunodiagnostic assay has been developed and approved by the FDA for use in the United States as a first tier assay. This test is based on the presence in a subject of antibodies to an immunodominant region (IR6) from the B. burgdorferi surface antigen, VlsE (VMP like sequence expressed), which is present in all three known pathogenic genospecies. The assay is described, e.g., in U.S. Pat. No. 6,475,492. The sequence of the peptide used in the approved assay, a 26 amino acid peptide derived from the European pathogenic Borrelai species, B garinii, is CMKKDDQIAAAMVLRGMAKDGQFALK (SEQ ID NO:2). An epitope mapping analysis of this peptide by the inventors of that patent (Liang et al. (2000) Infect Immun. 68, 2349-2353) concludes that, at least in monkeys and humans, this amino acid sequence is recognized as a single antigenic determinant.
The present inventors have identified a shorter version of the 26 amino acid IR6 peptide, which may contain certain amino acid substitutions, that provides the basis for a sensitive and specific immunoassay for Lyme disease.